A number of symptomologies which result in cognitive deficits, stroke, brain hemorrhage, and general mental debilitation appear to be associated with neuritic and cerebrovascular plaques in the brain containing the amyloid beta peptide (Aβ). Among these conditions are both pre-clinical and clinical Alzheimer's disease, Down's syndrome, and pre-clinical and clinical cerebral amyloid angiopathy (CAA). The amyloid plaques are formed from amyloid beta peptides. These peptides circulate in the blood and in the cerebrospinal fluid (CSF), typically in complexed form with lipoproteins. The Aβ peptide in circulating form is composed of 39–43 amino acids (mostly 40 or 42 amino acids) resulting from the cleavage of a common precursor protein, amyloid precursor protein, often designated APP. Some forms of soluble Aβ are themselves neurotoxic and may determine the severity of neurodegeneration and/or cognitive decline (McLean, C. A., et al., Ann. Neurol. (1999) 46:860–866; Lambert, M. P., et al. (1998) 95:6448–6453; Naslund, J., J. Am. Med. Assoc. (2000) 283:1571).
Evidence suggests that Aβ can be transported back and forth between brain and the blood (Ghersi-Egea, J-F., et al., J. Neurochem. (1996) 67:880–883; Zlokovic, B. V., et al, Biochem. Biophys. Res. Comm. (1993) 67:1034–1040; Shibata M, et al., J. Clin. Invest. (2000) 106:1489–1499). Further Aβ in plaques is in an equilibrium with soluble Aβ in the brain and blood (Kawarabayashi T, et al., J. Neurosci. (2001) 21:372–381).
As described in PCT application US00/35681 and U.S. Ser. No. 09/753,130 (now U.S. Pat. No. 6,465,195, issued Oct. 15, 2000) both incorporated herein by reference, total circulating levels of Aβ peptide in OSE are similar in normal individuals and individuals predisposed to exhibit the symptoms of Alzheimer's. However, Aβ42 levels are lower on average in individuals with Alzheimer's disease (Nitsch, R. M., et al., Ann. Neurol. (1995) 37:512–518). It is known that Aβ42 is more prone to aggregate than is Aβ40, and when this happens, adverse consequences such as Aβ deposition in amyloid plaques, conversion of Aβ to toxic soluble forms, nerve cell damage, and behavioral impairment such as dementias ensue (Golde, T. E., et al., Biochem. Biophys. Acta. (2000) 1502:172–187).
Methods to induce an immune response to reduce amyloid deposits are described in PCT publication WO99/27944 published Jun. 10, 1999. The description postulates that full-length aggregated Aβ peptide would be a useful immunogen. Administration of a Aβ fragment (amino acids 13–28) conjugated to sheep anti-mouse IgG caused no change in cortex amyloid burden, and only one in nine animals that received injections of the Aβ 13–28 fragment-conjugate showed any lymphoproliferation in response to Aβ40. The application also indicates that antibodies that specifically bind to Aβ peptide could be used as therapeutic agents. However, this appears to be speculation since the supporting data reflect protocols that involve active immunization using, for example, Aβ42. The peptides are supplied using adjuvants and antibody titers formed from the immunization, as well as levels of Aβ peptide and of the precursor peptide, are determined. The publication strongly suggests that Aβ plaque must be reduced in order to alleviate Alzheimer's symptoms, and that cell-mediated processes are required for successful reduction of Aβ plaque.
WO 99/60024, published 25 Nov. 1999, is directed to methods for amyloid removal using anti-amyloid antibodies. The mechanism, however, is stated to utilize the ability of anti-Aβ antibodies to bind to pre-formed amyloid deposits (i.e., plaques) and result in subsequent local microglial clearance of localized plaques. This mechanism was not proved in vivo. This publication further states that to be effective against Aβ plaques, anti-Aβ antibodies must gain access to the brain parenchyma and cross the blood brain barrier.
Several PCT applications that relate to attempts to control amyloid plaques were published on 7 Dec. 2000. WO 00/72880 describes significant reduction in plaque in cortex and hippocampus in a transgenic mouse model of Alzheimer's disease when treated using N-terminal fragments of Aβ peptides and antibodies that bind to them, but not when treated with the Aβ 13–28 fragment conjugated to sheep anti-mouse IgG or with an antibody against the 13–28 fragment, antibody 266. The N-terminal directed antibodies were asserted to cross the blood-brain barrier and to induce phagocytosis of amyloid plaques in in vitro studies.
WO 00/72876 has virtually the same disclosure as WO 00/72880 and is directed to immunization with the amyloid fibril components themselves.
WO 00/77178 describes antibodies that were designed to catalyze the hydrolysis of β-amyloid, including antibodies raised against a mixture of the phenylalanine statine transition compounds Cys-Aβ10-25, statine Phe19-Phe20 and Cys-Ap10-25 statine Phe20-Ala21 and antibodies raised against Aβ10-25 having a reduced amide bond between Phe19 and Phe20. This document mentions sequestering of Aβ, but this is speculation because it gives no evidence of such sequestering. Further, the document provides no in vivo evidence that administration of antibodies causes efflux of Aβ from the central nervous system, interferes with plaque formation, reduces plaque burden, forms complexes between the antibodies and Aβ in tissue samples, or affects cognition.
It has been shown that one pathway for Aβ metabolism is via transport from CNS to the plasma (Zlokovic, B. V., et al., Proc. Natl. Acad. Sci (USA) (1996) 93:4229–4234; Ghersi-Egea, J-F., et al., J. Neurochem. (1996) 67:880–883). Additionally, it has been shown that Aβ in plasma can cross the blood-brain-barrier and enter the brain (Zlokovic, B. V., et al., Biochem. Biophys. Res. Comm. (1993) 67:1034–1040). It has also been shown that administration of certain polyclonal and monoclonal Aβ antibodies decreases Aβ deposition in amyloid plaques in the APPV717F transgenic mouse model of Alzheimer's disease (Bard, F., et al., Nature Med. (2000) 6:916–919); however, this was said to be due to certain anti-Aβ antibodies crossing the blood-brain-barrier stimulating phagocytose of amyloid plaques by microglial cells. In Bard's experiments, assays of brain slices ex vivo showed that the presence of added Aβ antibody, along with exogenously added microglia, induced phagocytosis of Aβ, resulting in removal of Aβ deposits.
The levels of both soluble Aβ40 and Aβ42 in CSF and blood can readily be detected using standardized assays using antibodies directed against epitopes along the Aβ chain. Such assays have been reported, for example, in U.S. Pat. Nos. 5,766,846; 5,837,672; and 5,593,846. These patents describe the production of murine monoclonal antibodies to the central domain of the Aβ peptide, and these were reported to have epitopes around and including positions 16 and 17. Antibodies directed against the N-terminal region were described as well. Several monoclonal antibodies were asserted to immunoreact with positions 13–28 of the Aβ peptide; these did not bind to a peptide representing positions 17–28, thus, according to the cited patents, establishing that it is this region, including positions 16–17 (the α-secretase site) that was the target of these antibodies. Among antibodies known to bind between amino acids 13 and 28 of Aβ are mouse antibodies 266, 4G8, and 1C2.
We have now unexpectedly found that administration of the 266 antibody very quickly and almost completely restores cognition (object memory) in 24-month old hemizygous transgenic mice (APPV717F). Yet, the antibody does not have the properties that the art teaches are required for an antibody to be effective in treating Alzheimer's disease, Down's syndrome, and other conditions related to the Aβ peptide. To our further surprise, we observed that antibodies that bind Aβ between positions 13 and 28 (266 and 4G8) are capable of sequestering soluble forms of Aβ from their bound, circulating forms in the blood, and that peripheral administration of antibody 266 results in rapid efflux of relatively large quantities of Aβ peptide from the CNS into the plasma. This results in altered clearance of soluble Aβ, prevention of plaque formation, and, most surprisingly, improvement in cognition, even without necessarily reducing Aβ amyloid plaque burden, crossing the blood brain barrier to any significant extent, decorating plaque, activating cellular mechanisms, or binding with great affinity to aggregated Aβ.